The retinoid visual cycle refers to a multi-step pathway to recycle 11-cis retinal, a light sensitive chromophore for rod and cone visual pigments in vertebrates. A key step in the visual cycle is the conversion of all-trans retinyl ester to 11-cs retinol, which is catalyzed by an enzyme, namely isomerohydrolase. Previously, three groups, including ours, have independently reported that RPE65 has isomerohydrolase activity. Further, we have recently demonstrated robust isomerohydrolase activity in purified RPE65 protein reconstituted into liposomes, which provides conclusive evidence indicating that RPE65 is the isomerohydrolase in the visual cycle. Multiple recessive mutations in RPE65 have been shown to cause inherited retinal dystrophies, such as retinitis pigmentosa (RP) and Leber's congenital amaurosis. In these patient families, only individuals carrying mutations in both copies of the RPE65 gene manifest the disease phenotypes, as a single allele of the Wt RPE65 gene can generate sufficient isomerohydrolase to maintain the normal visual cycle. However, recent genetic studies have reported that some RP cases are caused by a dominant mutation of RPE65, in these cases; patients carrying the point mutation in a single allele of the RPE65 gene develop progressive vision loss. This represents a new pathogenic mechanism for RP caused by RPE65 mutations. In this study, we will investigate how this dominant RPE65 mutant interacts with the Wt RPE65 at the protein level and induces oligomerization and mislocalization of Wt RPE65, leading to impaired isomerohydrolase activity of Wt RPE65 and visual cycle deficiency. This study has potential to elucidate a novel pathogenic mechanism of RP caused by RPE65 mutations. A2E accumulation is a known pathogenic feature in Stargardt disease and AMD. Our recent study demonstrated that A2E binds with RPE65 and inhibits its isomerohydrolase activity, a possible new pathogenic mechanism by which A2E impairs vision. This project will use a Stargardt disease model to establish the inhibitory effects of endogenously generated A2E in the RPE on RPE65 activity and on the visual cycle. This study will provide in vivo evidence if the inhibition of RPE65 activity by A2E contributes to impaired vision in Stargardt disease and AMD. Oxidation of 11-cis retinol, the product of RPE65, to 11-cis retinal, the chromophore of visual pigments, is the final, yet important enzymatic reaction in the visual cycle. However, the enzyme catalyzing this reaction has not been identified and represents a missing component of the visual cycle. RDH10 is known to exist in the RPE65 complex and to convert 11-cis retinol to 11-cis retinal in vitro. This project will define the roleof RDH10 in catalyzing this reaction in the visual cycle using RPE-specific conditional RDH10 knockout mice. This study will define the function of RPE65 complex and fill a gap in the visual cycle.